Principle: The familiar "BioBrick cloning" enzymes (i.e., EcoRI, NotI, XbaI, SpeI, PstI) are Type II restriction enzymes, which cut the sequences that they specifically bind to. The Type IIS Assembly method uses a Type IIS restriction enzyme, which binds at a specific sequence and cuts at a non-specific location exactly five base pairs away. As a result, the enzyme cleaves away its own binding site and leaves behind the most useful feature of assembly, sticky overhangs. When designed properly, Type IIS sites can be used to perform seamless assembly of parts. As an added convenience, this protocol allows cutting and ligation to occur in a single tube, as a single reaction. Thus, gel purification steps can be eliminated.

First, map out your assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.

Design a pair of primers to add BsmBI sites to the ends of a vector backbone. The "cacacca" before BsmBI is used to help restriction enzyme positioning. The "a" after BsmBI is a spacer that is required to generate a correct 4-base sticky end.

Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bactial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).